JP2007530640A - Use of phenothiazine derivatives for preventing and / or treating hearing loss - Google Patents

Abstract

The present invention relates to a formula for the manufacture of a medicament intended to prevent and / or treat hearing loss.

[Wherein, R represents a hydrogen atom, an alkyl group, an arylalkyl group, or a —C (O) R ′ group]
Relating to the use of phenothiazine.

Description

  The present invention relates to the use of a phenothiazine derivative having calpain inhibitory activity and activity of scavenging reactive oxygen species (ROS) for the manufacture of a medicament intended to prevent and / or treat hearing loss. The invention also relates to products containing at least one such compound and pharmaceutical compositions containing it.

  Among many causes of hearing loss, diseases such as meningitis or otitis, genetic factors, trauma, tumors, drugs, medicines such as certain antibiotics, anticancer drugs, non-steroidal anti-inflammatory drugs, Mention may be made of diuretics, ulcer drugs or anticonvulsants, prolonged exposure to aromatic organic solvents such as toluene or xylene, aging and exposure to noise. Noise and senile deafness (deafness associated with aging) that have been exposed to medication for extended periods of time are a major cause of hearing loss.

  It is now well known that certain antibiotics of the aminoglycoside group, such as gentamicin or tobramycin (which are used to treat severe infections) are responsible for maze deafness. Toxicity to aminosides (aminoglucosides such as amikacin, dibekacin, gentamicin, isepamicin, netilmycin, spectinomycin, tobramycin) initially appears more frequently with hearing loss and is initially not recognized by the patient. The patient will be troubled by this gradually. Unfortunately, hearing loss is often irreversible.

  Noise around us reduces our hearing function. Hearing loss caused by noise occurs when the auditory hair cells that carry sound toward the inner ear are damaged and can no longer instruct the auditory nerve to send electrical pulses to the brain.

  Volume and duration of exposure are the two main factors affecting hearing loss. Although the response to exposure to noise varies from one person to the next, several factors can be reported with confidence. Studies have shown that long-term exposure to noise of 85 decibels (dB) or higher causes permanent hearing loss over time.

  European and North American statistics reveal that 8-10% of the population has cochlearism (deafness, hearing loss, tinnitus). Given the volume produced by discos, techno music concerts and walkmen, deaf people and people suffering from tinnitus are being produced in all generations. Thus, age-related problems (senile deafness) beginning recently at about 60 years of age begin even earlier, ie, at about 35-40 years old.

  A problem with auditory pathologies is that the majority is due to the loss of auditory hair cells and neurons in the inner ear (or cochlea). These cells pass the developmental stages that are essentially in the womb and do not have the ability to renew themselves after the final stage of differentiation.

  The progressive loss of ear sensation and neural capacity associated with various cochlear pathologies appears to be still beyond the scope of treatment today.

The subject of the invention is therefore a formula in the form of diastereoisomers or any combination of these isomers for the manufacture of a medicament intended to prevent and / or treat hearing loss.
[Wherein, R represents a hydrogen atom, a (C ₁ -C ₆ ) alkyl group, an arylalkyl group or a —C (O) R ′ group, wherein R ′ of the above group represents a heterocycloalkyl group, (C ₁ -C C ₆ ) represents an alkyl group, an aryl group or an aralkyl group;
However, the alkyl group, an aryl group or a heterocycloalkyl group, optionally (C ₁ ~C ₆₎ alkyl group, hydroxy group, (C ₁ ~C ₆₎ alkoxy group, a nitro group, a cyano group, a halogen atom or Optionally substituted with one or more of the same or different substituents selected from —NR ₁ R ₂ ; R ₁ and R ₂ are independently a hydrogen atom or a (C ₁ -C ₆ ) alkyl group Or R ₁ and R ₂ together with the nitrogen atom to which they are attached form an optionally substituted heterocycle]
Is the use of a heterocyclic derivative corresponding to

  As used herein, “trauma” refers to a group of local lesions including tissues and organs caused by external substances. In the case of acoustic trauma, the external material is mainly noise.

The (C ₁ -C ₆ ) alkyl group is a linear or branched alkyl group containing 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec- A butyl group and a tert-butyl group, a pentyl group, a neopentyl group, an isopentyl group, a hexyl group and an isohexyl group are meant. The (C ₁ -C ₆ ) alkoxy group can correspond to the aforementioned alkyl group, for example a methoxy group, an ethoxy group, a propyloxy group or an isopropyloxy group, or also a linear, secondary or tertiary butoxy group possible. The alkylcarbonyl group can correspond to the aforementioned alkyl group, and can be, for example, a methylcarbonyl group, an ethylcarbonyl group, or a propylcarbonyl group. A halogen atom means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

  An aryl group means a carbocyclic or heterocyclic system containing at least one aromatic ring, wherein the system is such that at least one of the rings constituting it is a heteroatom (O, N or S atom). ) Is called heterocyclic. Examples of the carbocyclic aryl group may include a phenyl group or a naphthyl group. Examples of heterocyclic aryl groups (heteroaryl groups) include thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, thiazolyl, isoxazolyl, oxazolyl, pyridyl, pyrazinyl, pyrimidyl Benzothienyl group, benzofuryl group and indolyl group.

  The term heterocycle (ie heterocycloalkyl) represents a saturated or unsaturated monocyclic or bicyclic heterocycle containing preferably 1 to 5 heteroatoms selected from O, N or S atoms. The nitrogen atom can be optionally substituted with a group selected from an alkyl group, an aryl group, an aralkyl group and an alkylcarbonyl group. Examples of saturated heterocycles include tetrahydrofuran, tetrahydropyran, oxetane, oxepane, tetrahydrothiophene, tetrahydrothiopyran, thietane, pyrrolidine, piperidine, azetidine, 1,3-dioxane, 1,3-dioxolane, 1,3-dithiolane, 1 1,3-dithiane, 1,3-oxathiolane, 1,3-oxazolidine, 1,3-imidazolidine or 1,3-thiazolidine. Examples of unsaturated heterocycles may include dihydrothiophene, dihydrofuran, dihydropyrrole, dihydroimidazole, dihydropyrazole, dihydropyridine, indoline.

  An arylalkyl (ie, aralkyl) group means a group in which each of the aryl group and the alkyl group has the above-mentioned meaning, for example, a benzyl group, a phenethyl group, or a naphthylmethyl group.

In the case of a group of the formula —NR ₁ R ₂ , wherein R ₁ and R ₂ together with the nitrogen atom to which they are attached form an optionally substituted heterocycle The heterocycle is preferably saturated and consists of 4 to 7 members and 1 to 3 hetero atoms including nitrogen atoms already present. Said additional heteroatoms are independently selected from the group constituted by O, N and S atoms. The heterocycle can be, for example, an azetidine ring, a pyrrolidine ring, a piperidine ring, a piperazine ring, a morpholine ring or a thiomorpholine ring. Said heterocycle can be substituted with one or more identical or different substituents selected from hydroxy, alkyl, aryl, aralkyl, alkoxy or halogen atoms.

  More particularly, the subject of the present invention is the use as described above, characterized in that R represents —C (O) R ′ and preferably R ′ represents an alkyl group.

Most preferably, compound (I) is characterized in that R represents —C (O) —CH ₃ . The latter compound will hereinafter be known as compound (I).

  More particularly, the subject of the present invention is the use as described above, characterized in that R represents a hydrogen atom.

Also very preferably, said compound (I) has the formula
For more details
Have one of these.

Also very preferably, said compound (I) has the formula
For more details
Have one of these.

  The aforementioned compound is a protective agent having both an antioxidant effect and an anti-calpain effect, and is described in International Application Publication No. WO01 / 32654.

  The subject of the present invention is therefore also the use of a compound of formula (I) above before or after treatment for the cause of hearing loss.

  The subject of the present invention is also the use of a compound of formula (I) as described above for the manufacture of a medicament intended to prevent and / or treat hearing loss after administration of another medicament. Preferably, said another medicament is an antibiotic such as gentamicin, an anticancer drug such as cisplatin, a non-steroidal anti-inflammatory agent such as salicylic acid derivative or ibuprofen, a diuretic such as furosemide, an anti-ulcer such as cimetidine or omeprazole Anticonvulsants such as drugs, carbamazepine or valproate. Said other medicament is very preferably an antibiotic, in particular gentamicin.

  The subject of the present invention is also the use of a compound of formula (I) as described above for the manufacture of a medicament intended to prevent and / or treat hearing loss after senile deafness.

  The subject of the present invention is also the use of a compound of formula (I) as defined above for the manufacture of a medicament intended to prevent and / or treat hearing loss after acoustic trauma.

  The compounds of the invention can be used alone or at least one other substance with pharmaceutical activity, preferably to prevent and / or treat hearing loss or to prevent and / or treat diseases associated with hearing loss. Alternatively, it can be used in combination with a substance that can be treated.

  The compounds include antioxidants, calpain inhibitors such as leupeptin or Neurodur, peripheral vasodilators such as EGb 761 (registered trademark), agonists or antagonists of NMDA receptors, c-Jun such as D-JNK-1. Can be combined with peptide inhibitors of N-terminal kinases.

  The subject of the present invention is also at least one further substance with pharmacological activity, preferably antioxidants, calpain inhibitors, peripheral vasodilators, agonists or antagonists of NMDA receptors, c-Jun N-terminal kinase Said use characterized in combination with a substance selected from the peptide inhibitors of

  The compound of formula (I), preferably the compound (1), can be administered at a dose of 50 to 500 μM for local application. In the case of local intracochlear treatment, the compound can be administered at a dose comprised between 50 and 200 μM. In the case of topical extracochlear treatment, the compound can be administered at a dose comprised between 200 and 500 μM. Substances, optionally in combination with them, known in pharmacology, are administered at standard recommended doses.

  The aforementioned compounds and optionally pharmaceutically active substances in combination therewith can be administered by standard routes of administration, for example oral, intraperitoneal, subcutaneous or intravenous routes. These substances can be administered simultaneously or separately by the same route of administration or by different routes of administration. Preferably, said compound (I) is topically administered in the usual manner for the treatment of the inner ear, for example in a tube equipped with a microcatheter, a transtymotic infusion syringe or a Silverstein Microwick type wick.

  The subject of the invention is also a diastereomeric form or a combination of these diastereomeric forms in the form of the above-mentioned heterocyclic derivatives of formula (I) and at least one substance having therapeutic activity, A product containing as a concomitant for use simultaneously, separately or over time to prevent and / or treat hearing loss. Preferably, the subject of the present invention is a product as described above for preventing and / or treating hearing loss after administration of a medicament or after senile deafness or after acoustic trauma.

  Finally, the subject of the present invention is the aforementioned product as a medicament.

  In the case of hearing loss after administration of the medicament, the medicament is preferably an antibiotic, preferably gentamicin.

  Substances having pharmacological activity, optionally combined, are administered by the route of administration normally envisaged for these substances in the therapeutic field under consideration.

  In the case of hearing loss caused by acoustic trauma, administration of said compound (I) may occur several days before acoustic trauma, preferably 2-3 days prior to trauma and 24 hours after trauma. Preferably, this administration occurs within 7 days after the acoustic trauma. Also preferably, this administration can occur within 2 hours after the acoustic trauma.

  The subject of the present invention is therefore the administration of said compound (1) in diastereomeric form or in any combination of these diastereomeric forms within 7 hours, preferably within 1 hour after trauma. Said use, characterized in that

  The results showing the therapeutic efficacy of compound (1) for functional recovery after acoustic trauma are presented in the experimental part.

  The following examples are given for the purpose of illustrating the above process and should not be considered as limiting the scope of the invention in any way.

Experimental part:
Pharmacological research
1) Demonstration of the protective effect of Compound 1 administered as a co-treatment against the loss of auditory hair cells induced by gentamicin induced toxic deafness induced by treatment with gentamicin Gentamicin and other aminoglycosides are It has been shown to cause damage to auditory hair cells and hearing loss. Zebrafish shows sensory organs called nerve hills on its body surface. In the case of this fish, the neurone auditory hair cells can be stained using DASPEI, which reflects the number of auditory hair cells. The auditory hair cells are structurally and functionally similar to the internal auditory hair cells of the human ear.

  Damage to internal auditory hair cells is caused by gentamicin in zebrafish. To test the effect of Compound (1) on the protection of auditory hair cells damaged by gentamicin, Compound (1) was administered as a co-therapeutic with gentamicin. The internal auditory hair cells were then stained and quantified.

  This study was performed on 5-day-old fish incubated for 24 hours with 1 μg / ml gentamicin in the presence or absence of compound (1). Controls were performed in parallel; single vehicle (1% DMSO; positive control). Fish treated with gentamicin is a negative control.

  DASPEI (2,4-dimethyl-aminostyryl-N-ethylpyridinium iodide) staining was performed to visualize auditory hair cells in vivo (n = 5 per group). Morphometric analysis was used to quantify the auditory hair cell staining signal. The positive control DASPEI staining signal was defined as 100%.

  The results obtained are shown in FIG. 1 (% of auditory hair cells stained with DASPEI). Positive control (zebrafish—1% DMSO); negative control (zebrafish—1% DMSO—gentamicin 1 μg / ml) and product effect (zebrafish—1% DMSO—gentamicin—compound 1). The experiment was performed on 5 animals per group.

The result of FIG.
The staining signal of the negative control corresponds to 31.5 ± 4.2% of the control signal, or 68.5 ± 4.9% loss of auditory hair cells after treatment with gentamicin,
-The staining signal of the animals treated with gentamicin and compound 1 corresponds to 65.2 ± 4.4% of the control signal, or 48.7 ± 2.63% of auditory hair cells damaged by treatment with gentamicin. This is equivalent to safeguarding.

2) Hearing loss after acoustic trauma This is a pre- and post-treatment compound after hearing loss caused by acoustic trauma in guinea pigs on the one hand protection of auditory hair cells in the inner ear and on the other hand auditory function recovery ( Including the study of the protective effect of 1). Compound (1) was administered by the “intracochlear or extracochlear” topical route to simulate its use in human surgical clinical medicine.

  The functional recovery obtained with this novel protective agent is quantified using a functional test that is an audiogram reading of the animal. This audiogram is created by recording the activity of possible synthetic actions of the auditory nerve. Audiograms are recorded before and after acoustic trauma. Analysis by scanning electron microscopy completes this functional data by studying cell loss along the cochlear helix and the protective effect of compound (1).

  All experiments were performed in guinea pigs: each experiment was performed according to a similar schema.

The general schema for the experimental phase is as follows:
The animals were anesthetized with an intramuscular injection of a mixture of Rompun® 2% (3 mg / kg) + Zoletil® (40 mg / kg). This anesthesia has the advantage of rapidly resolving and can be maintained for several hours by regular injections (every 2 hours) of one third of the initial dose.

The arrangement of the electrodes and minipumps of FIG.

  I approached the cochlea with access from the back. After shaving and cleaning the head, a 2 cm incision was made after the pinna. Lay the ear lower surface and muscle surface covering the tympanic vesicle. Immediately after drying and cleaning, a hole was drilled in the upper wall below where the facial nerve appeared in the bone. Then, an active electrode (platinum wire covered with Teflon and having a diameter of 0.13 mm) was introduced into the tympanocyst and placed in contact with the membrane of the round window under the control of a surgical microscope (WILD M650). After recording the metric auditory threshold, small holes with a diameter of 0.2 mm were conically spread with each other with the basal turn of the tympanic nerve tubule just below the round window.

  A glass pipette (0.1 mm diameter at the tip) connected to the micropump via a catheter was inserted into the cochlea using a second micromanipulator.

  The tympanic vesicle containing the recording electrode and perfusion pipette was closed with dental resin.

  The micropump was slid under the animal's skin, betadine was applied to the muscle and skin surfaces, sutured with absorbable sutures, and covered with an antibiotic solution [Rifocin 5P100®].

  Next, a second incision of about 1.5 cm was made in the crown to fix the connector (Connectral, ref .: 8 / 45-05.050.000) on the skull of the animal. After scrubbing the skull periosteum, the area was carefully dried, silver nitrate applied, and then covered with a film of cyanolite.

  The (active and reference) electrodes were slid under the skin to the connector to which they were joined. The connector was then secured to the skull using dental resin.

Stimulation method:
Sound stimulation was generated using two Hewlett Packard synthesizers (HP 3314 A and HP 8904 A) and transmitted in the free field using a round speaker (JBL 075) placed 10 cm away from the ear. The calibration of the sound device is a measurement amplifier that allows direct reading of the sound level of a 1/2 inch microphone (model 4134, Bruel and Kjaer) and decibel SP (dB SPL, reference: 2.10-5 Pa) in the cochlear implant. Model 2606) was used. In order to display the acoustic signal, the output of the measuring amplifier was connected to an oscilloscope. Animals were exposed to 6 kHz sound for 30 minutes at 120 dB SPL.

Recording method Amplify the cochlear reaction recorded by the electrode embedded in the cochlea via a connector fixed to the animal's head (amplification factor 1000) and use a preamplifier and GRASS P 511 K type differential amplifier And filtered (32 Hz to 3200 Hz). A direct trace was displayed on an oscilloscope (Tektronix type 513). This signal was averaged (256 passes) to reduce background noise and stored on a 486 PC computer, 66 megahertz (Hewlett-Packard-Vectra 05/65). The limit criterion was defined as the dB SPL value required to produce a measurable response (> 2 μV). Electrodes placed in contact with the cochlea (round window) allow the generation of cochlear responses and audiograms for each ear. The metered acoustic threshold was recorded 20 minutes after the acoustic trauma and recorded daily for 1 month.

Involvement of calpain activation after acoustic trauma:
For the purpose of measuring the activity of calpain enzyme after acoustic trauma, the cleavage of a specific substrate (fodrin) of calpain was quantified. Calpain cleaves 240 KD fodrine to produce a 150 KD degradation product. Double marking was performed using a polyclonal antibody specific for the 150 KD fragment and an anti-calbindin antibody that allows for the identification of auditory hair cells. Fluorescence was shown using a confocal microscope.

Molecular mechanism of death of auditory hair cells after acoustic trauma:
To investigate the nature of cell death, cochlear cell DNA fragmentation was quantified using the TUNEL method.

Measuring the integrity of auditory hair cells after acoustic trauma:
Cochlear cell integrity was examined by immunocytochemistry using anti-cytochrome C antibody. In healthy cells, cytochrome C is localized in mitochondria. After acoustic trauma, cytochrome C is diffused and distributed in the cytoplasm.

Phase I : Demonstration of protective effect of compound (1) administered as a pretreatment against hearing loss caused by trauma and cell extinction along the cochlear spiral:
Protocol-Phase 1: An osmotic minipump placed under the pre-dosed skin of Compound (1) delivers Compound (1).

  The animals were given acoustic trauma inside the cochlea with a catheter 2 days after the minipump was implanted (intracochlear perfusion).

  This experiment was performed on 7 animals followed by a dose response on 30 animals.

  Compound (1) was administered directly to the cochlea 2 days before trauma by an osmotic minipump (flow rate 1 μl / hour, volume 200 μl, diffusion period 7 days) permanently implanted in the cochlea. This method allows the measurement of the protective effect of compound (1) at a dose of 100 μM on cell loss and auditory function recovery along the cochlear spiral caused by trauma. In this case, the dose effect of the compound allows the determination of an effective dose that makes it possible to preserve 50% of the hearing.

Phase 1 results
Compound at a concentration of 100 μM (1)
Functional test measured with audiogram. Hearing loss after acoustic trauma and protection by Compound 1
On day 5 after 30 minutes of injury at 120 dB, the audiogram reading allowed to demonstrate the effectiveness of compound (1); this compound was perfused at 100 μM 2 days prior to this trauma. In some cases, 100% recovery of hearing was possible (Fig. 3).

Morphological study: histology of cells along the cochlear helix after acoustic trauma. Protection with Compound (1) At the end of the electrophysiological evaluation (audiogram), ie 30 days after acoustic trauma, animal cochleas were collected and prepared for electron microscopy.

  Cell loss was examined by counting tufted branches of the cilia using a scanning microscope. Quantitative data was obtained by performing high magnification observation of the surface of the Corti device.

  After 30 minutes of injury at 120 dB, the inner auditory hair cells were destroyed and some of the three rows of outer auditory hair cells were destroyed. The histology of these cells makes it possible to demonstrate the effectiveness of compound (1). This compound made it possible to protect 100% of the inner auditory hair cells and most of the outer auditory hair cells when perfused with 100 μM two days prior to this trauma (FIG. 4: histological data). Photo A: acoustic trauma; photo B: compound (1) + acoustic trauma; I = internal auditory hair cells; 0 = external auditory hair cells).

Involvement of calpain activation after acoustic trauma. Inhibition by Compound 1.

  The activation of the calpain enzyme was investigated by quantifying the degradation of the enzyme's specific substrate fodrine to a 150 KD fragment.

  This cleavage of fodrine was not detected in control cochlear cells.

  After acoustic trauma, immunomarking of the 150 KD fragment resulting from the cleavage of fodrine by activation of calpain enzyme was visible on external cochlear cells by marking with anti-FBDP antibody (“Hodolin-degrading organism”, green fluorescence; 5A: 48 hours after acoustic trauma). This activation of calpain activity after acoustic trauma is associated with the disappearance of cochlear cells visualized by the absence of marking with anti-calbindin antibodies that allow the identification of intact auditory hair cells.

  Topical administration of 100 μM compound (1) (a calpain activation inhibitor and an antioxidant) prevents fodoline cleavage by calpain enzymes in cochlear cells exposed to acoustic trauma. The absence of this green fluorescent marking, ie, the absence of non-degradation of the specific substrate of calpain, was associated with protection of cochlear cells (marking with anti-calbindin antibody; FIG. 5B) .

Molecular mechanism of death of auditory hair cells after acoustic trauma. Protection with compound 1.

  In order to investigate the nature of cochlear cell death caused by acoustic trauma, DNA fragmentation (quantified by the TUNEL method) was performed on animals undergoing acoustic trauma. The effect of Compound 1 was tested on this parameter to investigate its involvement by this apoptotic cell death mechanism.

  “TUNEL positive” cells were not found in cochleas obtained from control animals that were not exposed to noise.

  The cochlea exposed to acoustic trauma had TUNEL positive cell nuclei in the area of the Corti organ (FIG. 6A: 48 hours after acoustic trauma). Labeled nuclei were localized in the upper region of the Corti organ. Thus, these labeled nuclei appear to belong to auditory hair cells rather than feeder cells. A number of labeled nuclei could be seen 1 hour after the acoustic trauma, and this label could still be seen 4 days after the acoustic trauma. Thus, cell death of auditory hair cells after acoustic trauma is obtained by an apoptotic mechanism associated with DNA fragmentation.

  Topical administration of 100 μM compound (1) (calpain activation inhibitor and antioxidant) prevented the nuclear marking of auditory hair cells (TUNEL method, FIG. 6B: Compound (1) —48 hours after acoustic trauma ). Compound (1) suppressed cell death due to apoptosis caused by acoustic trauma.

Loss of auditory hair cell integrity after acoustic trauma. Protection with compound (1).

  In order to investigate the loss of integrity of auditory hair cells after acoustic trauma, cytochrome C diffusion by immunocytochemistry with the use of anti-cytochrome C antibodies was performed in animals that had undergone acoustic trauma.

  Compound (1) was tested for cytochrome C release from the mitochondrial compartment to the cytoplasmic compartment with the aim of examining its effect on loss of integrity of auditory hair cells following acoustic trauma.

  In the cochlea obtained from control animals that were not exposed to noise, cytochrome C is localized in mitochondria.

  Cochlea exposed to acoustic trauma showed cytochrome C markings that diffused and distributed in the cytoplasm (FIG. 7A).

  100 μM compound (1) (a calpain activation inhibitor and an antioxidant) prevented mitochondrial to cytoplasmic dispersion of auditory hair cells exposed to cytochrome C acoustic trauma and thus maintained cell integrity (FIG. 7B).

Compound (1) Dose Response The protective effect of Compound (1) was evaluated by varying the dose with the goal of defining an effective amount (ED ₅₀ ) that restores 50% of hearing loss. Eight groups of 5 animals were used, such as a group that received only artificial perilymph and a group that received Compound (1) 1 μM, 3 μM, 10 μM, 33 μM and 100 μM, ie 30 animals.

On day 5 after 120 dB, 30 minutes of trauma, audiogram readings determine the effective amount of compound (1) (ED ₅₀ = 3.61 μM) that allows 50% recovery of post-traumatic hearing loss. (Fig. 8).

Phase II : Demonstration of protective effect of compound (1) administered by extracochlear perfusion in post-treatment against cell loss and hearing loss along the cochlear spiral. Determination of the time that Compound (1) allows 50% recovery of hearing after trauma.

Post-administration of compound (1):
Preliminary studies have shown that cochleostomy performed after exposure further exacerbates the traumatic effects of sound. In this way, animals that are implanted after trauma will not recover, and animals that are not implanted will not recover. In order to eliminate trauma associated with cochleostomy, we have developed a non-traumatic method by administering the compound of the present invention directly into the round window (extracochlear). developed.

  An osmotic minipump was implanted under the skin, and compound (1) was delivered to the cochlea by catheter 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours or 24 hours after the animal suffered acoustic trauma (extracochlear) Perfusion).

  This experiment was performed on 30 animals.

  Compound (1) is added 48 hours before trauma of the middle ear (cochlear perfusion) by osmotic minipump (flow rate 1 μl / hour, volume 200 μl, diffusion period 7 days) or 1 hour, 3 hours, 6 hours, 12 hours or Administered 24 hours later. This minipump is permanently implanted in the middle ear and diffuses the compound directly into the round window.

These experiments allow the determination of the withdrawal period (DH) of compound (1), ie the maximum time that compound (1) can show while demonstrating a protective effect after trauma. The effective time (ET ₅₀ ) that allowed 50% recovery of hearing lost after trauma was also examined. This method also allowed measurement of the protective effect of Compound (1) at a dose of 300 μM against cell loss along the spiral cochlea caused by the trauma one month after the acoustic trauma.

Phase 2 results
Compound (1) at a concentration of 300 μM
Functional test: audiogram One hour after trauma, Compound (1) maintains 90% of hearing. The time that Compound (1) can show while maintaining 50% of hearing after trauma was determined to be 6-7 hours. The treatment window for compound (1) is 24 hours after 30 minutes of acoustic trauma at 120 dB. This means that in this model compound (1) is active for the first 24 hours after acoustic trauma (FIG. 9: audiogram performed 10 days after acoustic trauma).

Morphological study: Histology of cells along the cochlear spiral 1 month after acoustic trauma External cochlear perfusion of 300 μM compound (1), started 6 hours after acoustic trauma, is an auditory hair cell 1 month after acoustic trauma Is still protecting most of it. In fact, the contralateral side exposed to noise but not treated with compound (1) (in this case 86% of internal auditory hair cells and 62% of external auditory hair cells were damaged by acoustic disturbances) Only 32% of the internal auditory hair cells and 18% of the external auditory hair cells are not present in the area damaged by acoustic disturbances.

It is explanatory drawing which showed the protective effect of the compound 1 administered as a simultaneous treatment with respect to the loss | disappearance of the auditory hair cell induced by gentamicin. It is explanatory drawing which showed arrangement | positioning of an electrode and a minipump. It is an audiogram showing the protective effect of the compound (1) against the hearing loss after acoustic trauma. It is a scanning micrograph of internal auditory hair cells and external auditory hair cells after 120 minutes of trauma at 120 dB. I indicates internal auditory hair cells, and O indicates external auditory hair cells. 2 is a scanning micrograph of external cochlear cells after acoustic trauma. 2 is a scanning photomicrograph of external cochlear cells topically administered with 100 μM compound (1) after acoustic trauma. It is a scanning micrograph of an auditory hair cell after acoustic trauma. It is a scanning micrograph of the auditory hair cell of local administration of 100 micromol compound (1) after an acoustic trauma. 2 is a scanning micrograph of a cochlea exposed to acoustic trauma. 2 is a scanning micrograph of a cochlea with topical administration of 100 μM compound (1) after acoustic trauma. It is a dose response curve of a compound (1). An audiogram performed for 10 days after acoustic trauma.

Claims (29)

Formula in the form of diastereoisomers or any combination of these isomers for the manufacture of a medicament intended to prevent and / or treat hearing loss
[Wherein, R represents a hydrogen atom, a (C ₁ -C ₆ ) alkyl group, an arylalkyl group or a —C (O) R ′ group, wherein R ′ of the above group represents a heterocycloalkyl group, (C ₁ -C C ₆ ) represents an alkyl group, an aryl group or an aralkyl group;
However, the alkyl group, an aryl group or a heterocycloalkyl group, optionally (C ₁ ~C ₆₎ alkyl group, hydroxy group, (C ₁ ~C ₆₎ alkoxy group, a nitro group, a cyano group, a halogen atom or Optionally substituted with one or more of the same or different substituents selected from —NR ₁ R ₂ ; R ₁ and R ₂ are independently a hydrogen atom or a (C ₁ -C ₆ ) alkyl group Or R ₁ and R ₂ together with the nitrogen atom to which they are attached form an optionally substituted heterocycle]
Use of heterocyclic derivatives corresponding to   Use according to claim 1, characterized in that R represents -C (O) R '.   Use according to claim 2, characterized in that R 'represents an alkyl group. Use according to claim 3 in which R is equal to or representative of the -C (O) -CH _3.   Use according to claim 1, characterized in that R represents a hydrogen atom. Compound (I) has the formula
Use according to any one of the preceding claims, characterized in that Compound (I) has the formula
7. Use according to claim 6, characterized in that Compound (I) has the formula
7. Use according to claim 6, characterized in that Compound (I) has the formula
Use according to any one of claims 1 to 5, characterized in that Compound (I) has the formula
9. Use according to claim 8, characterized in that Compound (I) has the formula
9. Use according to claim 8, characterized in that   Use according to any one of the preceding claims, characterized in that the derivative (I) is administered before treatment.   Use according to any one of claims 1 to 11, characterized in that the derivative (I) is administered after treatment.   Use according to any one of the preceding claims for the manufacture of a medicament intended to prevent and / or treat hearing loss after the administration of the medicament.   15. Use according to claim 14 after administration of another medicament selected from antibiotics, anticancer drugs, non-steroidal anti-inflammatory drugs, diuretics, anti-ulcer drugs, anticonvulsants.   16. Use according to claim 15 after administration of an antibiotic.   Use according to claim 16, characterized in that the antibiotic is gentamicin amino.   14. Use according to any one of claims 1 to 13 for the manufacture of a medicament intended to prevent and / or treat hearing loss after senile deafness.   14. Use according to any one of claims 1 to 13 for the manufacture of a medicament intended to prevent and / or treat hearing loss after acoustic trauma.   20. Use according to claim 19, characterized in that compound (I) is administered within 7 hours, preferably within 1 hour after acoustic trauma.   Use according to any one of the preceding claims, characterized in that compound (I) is combined with at least one other substance having pharmaceutical activity.   Compound (I) is combined with at least one other substance having pharmaceutical activity capable of preventing and / or treating hearing loss or preventing and / or treating a disease associated with hearing loss Use according to any one of the preceding claims, characterized in that   That another substance having pharmacological activity is selected from an antioxidant, a calpain inhibitor, a peripheral vasodilator, an agonist or antagonist of NMDA receptor and a peptide inhibitor of c-Jun N-terminal kinase. 23. Use according to one of the claims 21 or 22, characterized.   Simultaneously for preventing and / or treating hearing loss, the heterocyclic derivative of formula (I) according to any one of claims 1 to 11 and at least one substance having therapeutic activity, A product that contains as a concomitant for use separately or over time.   25. A product according to claim 24 for preventing and / or treating hearing loss after administration of a medicament.   26. Product according to claim 25 for preventing and / or treating hearing loss after administration of an antibiotic, preferably gentamicin.   25. A product according to claim 24 for preventing and / or treating hearing loss after presbycusis.   25. A product according to claim 24 for preventing and / or treating hearing loss after acoustic trauma. 29. A product according to any one of claims 24-28 as a medicament.